An Alpha-Particle Emitting Antibody ([²¹³Bi]J591) for Radioimmunotherapy of Prostate Cancer¹

Michael R. McDevitt, Els Barendswaard, Dangshe Ma, Lawrence Lai, Michael J. Curcio, George Sgouros, Åse M. Ballangrud, Wei-Hong Yang, Ronald D. Finn, Virginia Pellegrini, Maurits W. Geerlings, Jr., Mona Lee, Martin W. Brechbiel, Neil H. Bander, Carlos Cordon-Cordo, and David A. Scheinberg²

Department of Pharmacology and Molecular Therapeutics [M.R.McD., E.B., D.M., M.J.C., L.L., V.P., M.L., and D.A.S.], Department of Radiology [R.D.F.], Department of Pathology (C. C.-C.) and Department of Medical Physics [G.S., Å.M.B., W.-H.Y.], Memorial Sloan-Kettering Cancer Center, New York, New York 10021; Department of Urology, [N.H.B.], New York Presbyterian Hospital-Weill Medical College of Cornell University and Ludwig Institute for Cancer Research, New York, New York 10021; Radioimmune and Inorganic Chemistry Section [M.W.B.], Radiation Oncology Branch, NCI, NIH, Bethesda, MD 20892; Pharmactinium, Inc. [M.W.G., Jr.], Chevy Chase, MD  20815. 

ABSTRACT

A novel alpha-particle emitting monoclonal antibody (mAb) construct targeting the external domain of Prostate Specific Membrane Antigen (PSMA) was prepared and evaluated in vitro and in vivo.  The chelating agent, N-[2-amino-3-(p-isothiocyanatophen-yl)propyl]-trans-cyclohexane-1,2-diamine-N,N’,N’,N’’,N’’-pentaacetic acid, was appended to J591 mAb to stably bind the ²¹³Bi radiometal ion.  Bismuth-213 is a short-lived (t_1/2 = 46 min.) radionuclide that emits high energy alpha-particles with an effective range of 0.07-0.10 mm which are ideally suited to treating single celled neoplasms and micrometastatic carcinomas.  The LNCaP prostate cancer cell line had an estimated 180,000 molecules of PSMA per cell; J591 bound to PSMA with a 3 nM affinity.  After binding, the radiolabeled construct-antigen complex was rapidly internalized into the cell, carrying the radiometal inside.  [²¹³Bi]J591 was specifically cytotoxic to LNCaP.  The LD₅₀ value of [²¹³Bi]J591 was 220 nCi/mL at a specific activity of 6.4 Ci/g.  The potency and specificity of [²¹³Bi]J591 directed against LNCaP spheroids, an in vitro model for micrometastatic cancer, also was investigated.  [²¹³Bi]J591 effectively stopped growth of LNCaP spheroids relative to an equivalent dose of the irrelevant control [²¹³Bi]HuM195 or unlabeled J591.  Cytotoxicity experiments in vivo were carried-out in an athymic nude mouse model with an i.m. xenograft of LNCaP cells.  [²¹³Bi]J591 was able to significantly improve (P<0.0031) median tumor-free survival (54 d) in these experiments relative to treatment with irrelevant control [²¹³Bi]HuM195 (33 d), or no treatment (31 d).  PSA was also specifically reduced in treated animals.  At day 51 mean PSA values were 104 ng/mL +/- 54 ng/mL (n=4, untreated animals), 66 ng/mL +/- 16 ng/mL (n=6, animals treated with [²¹³Bi]HuM195), and 28 ng/mL +/- 22 ng/mL (n=6, animals treated with [²¹³Bi]J591).  The reduction of PSA levels in mice treated with [²¹³Bi]J591 relative to mice treated with [²¹³Bi]HuM195 and untreated control animals was significant with P<0.007 and P<0.0136, respectively.  In conclusion, a novel [²¹³Bi]-radiolabeled J591 has been constructed which selectively delivers alpha-particles to prostate cancer cells for potent and specific killing in vitro and in vivo. 

INTRODUCTION

Metastatic prostate cancer that is resistant to hormone therapy has been treated with non-specific, systemic bone-seeking agents such as ³²P, ⁸⁹Sr, and ¹⁵³Sm (1-3) having the palliation of pain as the objective endpoint.  Specifically targeted radiotherapy using prostate tumor specific CC49 antibody labeled with ¹³¹I has been evaluated in two clinical trials with patients having metastatic prostate carcinoma.  In one trial, antibody localization was seen in 11/14 patients along with significant myelosuppression, however, it was estimated that there were low radiation doses to tumor (approx. 1,000 cGy); there was no repeat dosing due to HAMA responses and no patients met the radiographic or PSA criteria for objective response (4).  In the second trial, a high degree of tumor localization was observed in 13/15 patients, but HAMA response was seen in all patients; some degree of anti-tumor effect, as measured by pain relief, was seen in 6/10 symptomatic patients.  However, once again, no patients met the radiographic or PSA criteria for objective response (5).

Several mAbs that target prostate cancer were modified with the N-[2-amino-3-(p-isothiocyanatophenyl)propyl]-trans-cyclohexane-1,2-diamine-N,N’,N’,N’’,N’’-pentaacetic acid (SCN-CHXA”-DTPA) moiety, labeled with ²¹³Bi and their in vitro potency towards cancer cell lines evaluated (6).  This comparative study of potential therapeutic anti-prostate mAbs resulted in the selection of J591 as the IgG to study further.  J591 targets the external domain of PSMA (7-9).  PSMA is a highly restricted prostate epithelial cell integral membrane glycoprotein, providing an immunogenic extracellular domain.  PSMA is expressed by a high proportion of prostate carcinomas and its expression is further increased in higher grade cancers, in metastatic disease and in hormone-refractory prostate cancers.  There are several mAbs targeting the external domain of PSMA which have been developed and described in the literature (7-9).

The construction of an alpha-particle emitting IgG radiotherapeutic moiety represents a “new class” of potent and specific radiopharmaceuticals used only recently in humans (10,11).  The radionuclide ²¹³Bi is a short-lived (t_1/2 = 46 min) a-particle emitting metal-ion generated from the decay of Ac-225.  Bismuth labeled HuM195, an anti-CD33 antibody, demonstrated specific and potent cell killing ability when directed against a leukemia cell line (12).  A robust, reliable ²²⁵Ac/ ²¹³Bi generator system was developed at MSKCC (13,14) and used to treat 18 patients with 80 doses of [²¹³Bi]HuM195 in a Phase I clinical trial (11).  Clinical conditions for reproducible radiolabeling of antibodies and quality assurance testing were developed (15).  Feasibility for human use and therapeutic activity were demonstrated.  The low abundance, low energy g-emmissions associated with the decay of ²¹³Bi (440 KeV, frequency of 16% per disintegration) were utilized in evaluating the biodistribution patterns of [²¹³Bi]HuM195 in humans and in performing dosimetry calculations (16-18). 

Alpha-particle therapy has been proposed for use in single cell disorders, such as leukemias.  The purpose of the current study was to evaluate the biochemistry, biology, and cytotoxicity of a potent [²¹³Bi]-labeled J591 construct in a series of in vitro and in vivo models of prostate cancer to determine whether radioimmunotherapy with alpha-particles also would be suitable for clinical use against prostate cancer.  Such an approach would be particularly appropriate after debulking radiation or surgery in order to reduce the size of metastatic deposits of cancer. 

RESULTS

Equilibrium Binding Affinity and the Number of Antigen Binding Sites

            A nonlinear regression curve fit analysis (R² = 0.995) of the [¹¹¹In]J591 equilibrium binding data yielded an immunoreactivity corrected B_max value of 13,744 cpm per 50,000 LNCaP cells (Figure 1).  Using this value of B_max it was estimated that there were 180,000 molecules of J591 bound per cell.  The equilibrium binding affinity, Kd, was taken from the fit curve at 0.5 times B_max and estimated to be 3 nM. 

Modulation of Cell Surface Antibody-Antigen Complexes

            The [¹¹¹In]J591-PSMA (cell surface antibody-antigen) complex was rapidly internalized immediately after binding to the cell.  Approximately 66% of the complex was internalized after 2 h and 79% internalized after 4 h (Figure 2).  The total cell associated counts and therefore, the cell surface counts continued to increase as a function of time through 24 h. 

Cell Kill Against Single Cells in vitro

LNCaP cell kill by [²¹³Bi]J591 proved to be both specific activity dependent and activity concentration dependent (Figure 3).  The LD₅₀ value of [²¹³Bi]J591 was 220 nCi/mL at a specific activity of 6.4 Ci/g ; 315 nCi/mL at a specific activity of 3.8 Ci/g; 4,400 nCi/mL at a specific activity approaching 0.06 Ci/g.  At the low specific activities represented by the last group, there is essentially no specific binding of the alpha-particle emitting J591 to the targets.  Thus, this level of cell kill represents the non-specific cytoxicity in the system used here.  Additional experiments evaluated [²¹³Bi]J591 against a PSMA negative tumor cell line, SKOV3.  The LD₅₀ value derived was 13,000 nCi/mL at a specific activity level of 1.6 Ci/g (not shown).  Therefore, cytotoxicity was specific activity-related, dose-related, and antigen specificity-dependant. 

Cell Kill Against Multicellular Spheroids in vitro

We have also investigated the potency and specificity of [²¹³Bi]J591 directed against LNCaP spheroids by assessing the change in volume of the spheroid as a function of time following various treatments.  The spheroids initially were comprised of approximately 1,000  LNCaP cells.  The volumes of all of the spheroids treated with a single dose of [²¹³Bi]J591 decreased markedly over a 2 month period (Figure 4).  Their size was more than 100-fold smaller than the untreated spheroid control group in this time period.  The [²¹³Bi]HuM195 treated spheroids demonstrate a 1 week delay in growth, followed by an increase in volume.  Two other control groups, untreated spheroids and spheroids exposed to unlabeled J591 (0.027 mg/mL), showed similar increases in volume to each other as a function of time with an almost 100-fold increase in spheroid volume after 1 month. 

LNCaP Tumor Model in Mice

            LNCaP tumor cells were mixed with Matrigel (Becton Dickinson Labware, Bedford MA) and xenografted into 8 week old athymic nude mice.  Mice received an i.m. injection of 6-7E6 LNCaP tumor cells mixed with Matrigel in the right hind leg in a volume of 0.25 mL.  Tumor growth in vivo was assessed histologically at days 2, 3, 5, 7, and 10 (Table 1).  At the time RIT was administered (day 2), the tumors were characterized histologically as disorganized cell clusters and nodules each comprised of several thousands of cells.  The nodules were not vascularized and not encapsulated.  On day 3,  the tumors were more organized and were becoming vascularized, but still not encapsulated.  By the fifth day, vascularization was more pronounced and on day 7 the tumors were encapsulated.  The purpose of the RIT studies (described below) was to examine the ability of the agent to treat the tumors prior to encapsulation and neovascularization.      

Cell Kill in vivo 

A single course of the [²¹³Bi]J591 drug, administered in four daily doses, improved (P<0.0031) median tumor-free survival (Figure 5) of LNCaP xenografted mice relative to mice treated with [²¹³Bi]HuM195 or untreated controls.  The median tumor-free survival times were 31d (n=4, untreated animals), 33 d (n=6, [²¹³Bi]HuM195 treated animals), and 54 d (n=6, [²¹³Bi]J591 treated animals). 

PSA is an important surrogate marker for prostate cancer burden in humans (28).  It can also be used in mice with prostate cancer cell xenografts (29).  PSA levels in tumor bearing mice also responded to the treatment (Table 2).  Mean PSA values, 51 days after treatment, were 104 ng/mL +/- 54 ng/mL (n=4, untreated animals), 66 ng/mL +/- 16 ng/mL (n=6, [²¹³Bi]HuM195 treated animals), and 28 ng/mL +/- 22 (n=6, [²¹³Bi]J591 treated animals).  The reduction of PSA levels in mice treated with [²¹³Bi]J591 relative to mice treated with [²¹³Bi]HuM195 and untreated control animals was significant with P<0.007 and P<0.0136, respectively.  In another similar experiment where unlabeled J591 was also examined as an additional control, the mean PSA values 30 days after treatment were 31 ng/mL +/- 20 ng/mL (n=5, untreated animals), 36 ng/mL +/- 38 ng/mL (n=5, 0.02 mg J591 treated animals), 26 ng/mL +/- 21 ng/mL (n=10, [²¹³Bi]HuM195 treated animals), and 12 ng/mL +/- 8 ng/mL (n=12, [²¹³Bi]J591 treated animals) (Figure 6).  In this experiment, animals received either one single ²¹³Bi drug administration or four consecutive daily administrations of a smaller dose of drug.  There were no statistically significant differences in response (measured PSA levels) observed between the 1x daily and the 4x daily treatment regimens for the [²¹³Bi]J591 and the [²¹³Bi]HuM195 treatments, nor between the unlabeled J591 and untreated controls.  Reduction of PSA levels, however, in all mice (n=12) treated with [²¹³Bi]J591 (1x daily and the 4x daily treatment regimens pooled) relative to all mice (n=10) treated with [²¹³Bi]HuM195 (1x daily and the 4x daily treatment regimens pooled) and all control animals (groups untreated and treated with unlabeled J591 pooled) (n=10) was significant with P<0.0443 and P<0.0192, respectively.

DISCUSSION

Alpha-particle emitting radiolabeled antibody constructs have been proposed as potent, selective agents to kill single cells (30,31).  We have shown this in vitro and in humans using an alpha-particle emitting anti-CD33 mAb for treatment of leukemia (10-12,15,16).  Tens of billions to hundreds of billions of individual leukemia cells in the blood and marrow were killed safely.  The kinetics and geometry of single-cell killing, however, might not be predictive of killing micrometastatic clusters of tumor cells as would be expected with the early spread of carcinomas.  Therefore, we now ask whether an alpha-particle emitting agent would be useful in a model for solid tumors, such as prostate cancer, that form micrometastatic disease in the bone marrow.  In this report, we describe for the first time, the construction of a prostate-specific, alpha-particle emitting agent capable of: 1) binding and internalizing into target cells; 2) selectively killing both individual cells and 1,000-cell spheroid clusters; 3) prolonging tumor-free survival and reducing PSA in mice bearing prostate cancer xenografts. 

LNCaP cell kill in vitro using [²¹³Bi]J591 proved to be both specific activity dependent and activity concentration dependent.  There was a 20-fold difference in the LD₅₀ values in vitro, spanning a 100-fold range in specific activities, indicating the specificity of the drug for cells expressing PSMA.  Additional data demonstrated that [²¹³Bi]J591 did not specifically kill a PSMA negative tumor cell line, SKOV3, supporting the fact that the drug does not target or destroy tissue that does not express PSMA.  Therefore, [²¹³Bi]J591 was an effective and specific radiolabeled agent for ablation of individual prostate cancer cells in vitro.  This is an advantage of using alpha-particle emitting radionuclides, as the particles transfer their energy in a region that is in very close proximity to the target. 

We have also investigated the potency and specificity of [²¹³Bi]J591 directed against LNCaP spheroids initially comprised of approximately 1,000 cells in vitro.  Spheroids of this size approximate the micrometastatic disease condition and can serve as a therapeutic test for investigating the potency and specificity of alpha-particle emitting IgG directed against multicellular targets.  The [²¹³Bi]J591 must be efficacious against the spheroids if it is expected to be clinically effective against metastatic prostate cancer.  A single dose of the [²¹³Bi]J591 drug was effective in arresting the growth of LNCaP spheroid cells relative to an equivalent dose of [²¹³Bi]HuM195, an irrelevant control mAb which has a minimal effect against the LNCaP spheroids, a similar dose of unlabeled J591, and untreated spheroids. Control experiments exhibited similar spheroid growth kinetics with the average spheroid volume increasing almost 100-fold over a period of 1 month.  The [²¹³Bi]HuM195 treated spheroids exhibited a delay in growth of approximately 1 week, presumably due to nonspecific irradiation effects.  It was estimated that random hits from alpha particles emitted from nonspecific IgG dispersed in the media could result in delivery of about 1% of the radiation dose as compared to a [²¹³Bi]-labeled specific mAb (27).  Other experiments (Sgouros and Ballangrud, unpublished results) have shown that anti-PSMA IgG penetrates 1-2 cell layers into spheroids during the time period (45-90 min.) that ²¹³Bi would be expected to be most active.  The [²¹³Bi]J591 treated spheroids decreased in volume by about 10% over a 3 week period with no further growth during the 2 month duration of the experiment.  These data were repeated in a second experiment with similar results.  Since the spheroid model may resemble the micrometastatic disease situation, it was apparent that [²¹³Bi]J591, despite incomplete penetration into the spheroid mass, was able to specifically target large cell clusters, destroy targeted cells, and interrupt further growth of the unirradiated spheroid core.  Further studies ongoing in this area are focusing on the utility of a multiple dosing schedule that would effectively peel away the remaining cell layers that might still be viable.  A multiple dosing schedule may eradicate the disease point source in its entirety while targeting any other remaining or new disease sites; however the schedule and dose details need to be better understood. 

The two therapeutic experiments carried-out in vivo demonstrated the ability of [²¹³Bi]J591 to improve the duration of tumor-free survival and to suppress PSA relative to controls in an animal model.  Following the trends observed in the spheroid cytotoxicity experiments, unlabeled J591 was ineffective in treatment and was comparable to no treatment.  In addition, the [²¹³Bi]HuM195 demonstrated a minimal effect in delaying tumor growth (2 to 3 d) and in decreasing PSA values relative to controls.  Table II compares the P values for the observed PSA values between the [²¹³Bi]J591 treated mice and the [²¹³Bi]HuM195 treated mice and controls.  The tumor model employed in this study was not optimal for examining the efficacy of an alpha-particle emitting radioimmunotherapeutic agent since an alpha-particle has a pathlength of 2-4 cell diameters.  However, in the absence of reliable metastatic models for prostate cancer in vivo, we employed an i.m. tumor model and treated at a relatively early time point following tumor inoculation; this allowed us to observe an effect due to the [²¹³Bi]J591 agent that resulted in tumor growth delay and a lower PSA value relative to controls.  The specific [²¹³Bi]J591 was directed against clusters of cells that at the time of RIT were approximately the size of the spheroids which were studied in vitro.  As with the spheroids, the tumors in vivo were comprised of nodules containing 1-2 thousand LNCaP cells that were not vascularized and not encapsulated.  

We have been successful in a Phase I clinical trial using [²¹³Bi]HuM195 (10,11) to treat 18 patients with acute myelogenous leukemia.  Ten of twelve evaluable patients had reductions in peripheral blood leukemia cells, and 12 of 18 had decreases in bone marrow blasts.  Doses up to 1 mCi/kg were used safely and there was no acute toxicity seen.  In the treatment of leukemia there may be approximately 1 kg of tumor present (1E12 cells) with approximately 20,000 HuM195 molecules bound by individual HL60 leukemia cells (32). 

The clinical situation in prostate cancer may favor the use of a [²¹³Bi]-labeled antibody based upon the following: 1) low tumor burden, 2) anti-PSMA-IgG-PSMA internalization, and 3) the relatively large number of PSMA binding sites per cell.  In comparison to leukemia, there should be at least ten to one hundred times less prostate cancer tumor burden at the time of treatment, even with widespread micrometastatic lesions.  Furthermore, J591 results in the internalization of the J591-PSMA complex into LNCaP cells (8) which favors the therapeutic use of [²¹³Bi]-labeled IgG constructs as internalized radiometal ions 1) will tend to remain with the cell and not be released and carried away from the target and 2) decays within the cell will necessarily deposit energy within the cell.  We undertook the measurement of the radiometal-labeled construct-antigen complex internalization to determine the amount of drug internalized initially and as a function of time.  Cellular internalization of alpha-particle emitting radionuclides insures that the ²¹³Bi particulate decay traverses the cell, enhancing the cytotoxicity relative to surface bound species.  We anticipate the ability to internalize much of the targeted, cell bound [²¹³Bi]J591 activity within a 4 h period allowing the ²¹³Bi to decay and deliver its dose efficiently within the target cell.  Additionally, it has been observed that the LNCaP cells continue to bind J591 and internalize it following exposure to the construct, presumably by expression of new (recycled) antigen binding sites (8 and this work).  From a therapeutic standpoint, this modulation phenomenon works to therapeutic advantage as [²¹³Bi]J591 in circulation can continue to be bound even after initial saturation of the cell binding sites.  The LNCaP cells bind approximately 180,000 molecules of J591 which was about a 10-fold greater number of binding sites than the number of CD33 sites per leukemia cell.  Assuming that prostate cancer cells express similar levels of PSMA in vivo, typical therapeutic specific activities of [²¹³Bi]J591 (10 Ci/g) would result in delivery of approximately 80 ²¹³Bi-atoms per cell.  It has been demonstrated (12) that several ²¹³Bi-atoms targeted per cell may result in a lethal dose of alpha particles to half of these cells. 

In summary, the radioimmunopharmaceutical described in this study, [²¹³Bi]J591, has been demonstrated to potently and specifically eradicate individual prostate cancer cells and 1,000 cell spheroid clusters in vitro, and inhibit tumor growth and suppress PSA in vivo in animal models.  These features support the investigation of such an agent further in the human clinical setting to treat metastatic prostate cancer. 

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¹This work was supported by the CaPCure Foundation and NIH RO1 CA55349.  We thank Dr. R. Molinet and Dr. C. Apostilides (Institute for Transuranium Elements) for the supply of Ac-225 to support this work.  We would also like to thank Dr. Javier Pinilla (MSKCC) and Dr. Peter Smith-Jones (New York Presbyterian Hospital-Weill Medical College of Cornell University) for several stimulating discussions relevant to this project.  Dr. Scheinberg is a Doris Duke Distinguished Clinical Science Professor. 

²To whom requests for reprints should be addressed, at Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021.